Work done in this laboratory has led us to believe that factor XIa has proteolytic activity towards factor VIII. We initiated a series of experiments to determine the nature of this activity and its effect on the cofactor function of factor VIII in coagulation. Incubation of 100 ng of recombinant factor VIII with either 2.75 microg or 0.275 microg of purified human factor XIa resulted in the appearance on immunoblot of bands of approximately 48 kDa and 43 kDa. Addition of Ca++ and phospholipid appeared to make no contribution to this activity. Assay of the incubation mixture up to three hours following the initiation of the reaction demonstrated a gradual loss of factor VIII activity to about 50% of the starting activity. Incubation of 100 ng factor VIII that had been treated with 0.06 U of bovine thrombin with 2.75 microg factor XIa resulted in a decrease of factor VIII activity to about 5% of the initial activity within fifteen minutes. Addition of phospholipid to the mixture protected factor VIIIa from XIa mediated inactivation. Thrombin alone inactivated the factor VIII at about the same rate as factor XIa. Thrombin-activated factor VIII to which hirudin had been added to inhibit further thrombin activity spontaneously degraded at a much lower rate. Work is in progress to characterize the factor XIa cleavage sites on factor VIII. The rapidity of XIa action on VIIIa in the absence of phospholipid suggests that factor XIa may play an important role in controlling the activation of the "intrinsic" clotting system and in localizing it to membrane surfaces.